Patient-derived Xenograft Tumor Cells Research Articles

Primary intracranial sarcoma associated with DICER1 mutant: a case report and preclinical investigation.

Primary intracranial sarcoma (PIS) is a rare and aggressive pediatric brain tumor, which is partially associated with DICER1 mutant. Although the molecular genetic characteristics of this tumor have previously been investigated, novel therapeutic targets remain unclear. Further, the lack of faithful preclinical models has hampered the development of novel therapeutic strategies. Herein, we describe a pediatric case of PIS with DICER1 mutant and describe the development of the first novel patient-derived xenograft (PDX) model of this rare tumor. Somatic genomic profiling of the tumor revealed mutations in DICER1, TP53, and ATRX. Germline analysis further revealed a pathogenic variant of DICER1, significant for the diagnosis and management of hereditary tumor predisposition syndrome. Overall, we demonstrated that the PDX model faithfully retained the phenotype and genotype of the patient's tumor, as well as the DNA methylation profile. Through high-throughput drug screening using PDX tumor cells, we found that activation of the retinoic acid receptor (RAR) signaling pathway reduced tumor cell viability. These findings indicate that the RAR signaling pathway is a potential therapeutic target for PIS in DICER1 mutant.

CAR memory-like NK cells targeting the membrane proximal domain of mesothelin demonstrate promising activity in ovarian cancer.

Epithelial ovarian cancer (EOC) remains one of the most lethal gynecological cancers. Cytokine-induced memory-like (CIML) natural killer (NK) cells have shown promising results in preclinical and early-phase clinical trials. In the current study, CIML NK cells demonstrated superior antitumor responses against a panel of EOC cell lines, increased expression of activation receptors, and up-regulation of genes involved in cell cycle/proliferation and down-regulation of inhibitory/suppressive genes. CIML NK cells transduced with a chimeric antigen receptor (CAR) targeting the membrane-proximal domain of mesothelin (MSLN) further improved the antitumor responses against MSLN-expressing EOC cells and patient-derived xenograft tumor cells. CAR arming of the CIML NK cells subtanstially reduced their dysfunction in patient-derived ascites fluid with transcriptomic changes related to altered metabolism and tonic signaling as potential mechanisms. Lastly, the adoptive transfer of MSLN-CAR CIML NK cells demonstrated remarkable inhibition of tumor growth and prevented metastatic spread in xenograft mice, supporting their potential as an effective therapeutic strategy in EOC.

STEM-07. CONSERVATION AND FAITHFUL REPRESENTATION OF CIRCULAR EXTRACHROMOSOMAL DNA IN ORTHOTOPIC PATIENT-DERIVED MEDULLOBLASTOMA XENOGRAFTS

Abstract BACKGROUND Extrachromosomal DNA (ecDNA) drives tumor heterogeneity and correlates with poor survival across various cancer types. Patient-derived xenograft (PDX) models facilitate preclinical drug testing in vivo, enabling tumor growth analysis and molecular tumor profiling. These models, combined with single-cell profiling, offer insights into intratumoral heterogeneity under therapeutic pressure and the emergence of drug resistance. We investigate the abundance and conservation of ecDNA in PDX models to assess the faithful representation of ecDNA compared to their corresponding primary tumors. METHODS Whole-genome sequencing (WGS) was applied to 27 medulloblastoma (MB) PDX models and their tumors of origin using Amplicon Architect. ecDNA frequency in these models was also compared to that in the MB patient population. Optical genome mapping enabled accurate and reliable ecDNA sequence reconstructions. Multiome single-cell RNA and ATAC-sequencing of a PDX tumor enabled analysis of ecDNA conservation and intratumoral heterogeneity compared to similar data previously obtained from the primary tumor. RESULTS ecDNA is largely conserved in PDX models, emphasizing their relevance as faithful models of the primary tumors. MB PDX models exhibit significant higher ecDNA frequency (16 out of 27 tumors) as compared to their frequency in the overall patient population (~18%), suggesting ecDNA as a driver for successful establishment of MB PDX models. Single-cell analysis revealed that ecDNA is omnipresent in almost all PDX tumor cells, contrasting with only ~9% of tumor cells in the primary tumor. CONCLUSIONS ecDNA analysis in medulloblastoma primary tumors and in PDX models revealed that ecDNA is preserved in PDX models. These findings emphasize the relevance of PDX models in preclinical in vivo studies aimed at analyzing the role of ecDNA under therapeutic pressure and for analyzing clonal selection along with molecular evolution of ecDNA during the development of therapeutic resistance.

Receptor tyrosine kinase inhibition leads to regression of acral melanoma by targeting the tumor microenvironment.

Acral melanoma (AM) is an aggressive melanoma variant that arises from palmar, plantar, and nail unit melanocytes. Compared to non-acral cutaneous melanoma (CM), AM is biologically distinct, has an equal incidence across genetic ancestries, typically presents in advanced stage disease, is less responsive to therapy, and has an overall worse prognosis. Independent analysis of published genomic and transcriptomic sequencing identified that receptor tyrosine kinase (RTK) ligands and adapter proteins are frequently amplified, translocated, and/or overexpressed in AM. To target these unique genetic changes, a zebrafish acral melanoma model was exposed to a panel of narrow and broad spectrum multi-RTK inhibitors, revealing that dual FGFR/VEGFR inhibitors decrease acral-analogous melanocyte proliferation and migration. The potent pan-FGFR/VEGFR inhibitor, Lenvatinib, uniformly induces tumor regression in AM patient-derived xenograft (PDX) tumors but only slows tumor growth in CM models. Unlike other multi-RTK inhibitors, Lenvatinib is not directly cytotoxic to dissociated AM PDX tumor cells and instead disrupts tumor architecture and vascular networks. Considering the great difficulty in establishing AM cell culture lines, these findings suggest that AM may be more sensitive to microenvironment perturbations than CM. In conclusion, dual FGFR/VEGFR inhibition may be a viable therapeutic strategy that targets the unique biology of AM.

Abstract 2271: Conservation and faithful representation of circular extrachromosomal DNA in orthotopic patient-derived medulloblastoma xenografts

Abstract Background: Extrachromosomal DNA (ecDNA) is a driver of tumor heterogeneity and is associated with poor survival in many different adult and childhood cancer types. Patient-derived xenograft (PDX) models facilitate preclinical drug testing in an in vivo environment that enables analysis of tumor growth and survival and molecular tumor profiling. These models, combined with single-cell profiling, can be used to study intratumoral heterogeneity under therapeutic pressure and the emergence of drug resistance. Here we investigate the abundance and conservation of ecDNA in PDX models with the aim of assessing the faithful representation of ecDNA compared to the tumors from which they are derived. Methods: Whole-genome sequencing (WGS) was applied to 27 medulloblastoma (MB) PDX models and their tumors of origin using Amplicon Architect, a tool used to reconstruct focally amplified regions of DNA to provide insights into the landscape of cancer genomes. The frequency of ecDNA in these models was also compared to that of ecDNA in the MB patient population. Optical genome mapping enabled accurate and reliable ecDNA sequence reconstructions. Multiome single-cell RNA and ATAC-sequencing of a PDX tumor enabled analysis of ecDNA conservation and intratumoral heterogeneity compared to similar data previously obtained from the primary tumor. Results: Comparative analyses revealed that ecDNA is largely conserved in PDX models, emphasizing their relevance as faithful models of the primary tumors. In addition, we find that ecDNA is significantly more frequent in MB PDX models (16 out of 27 analyzed patient-derived tumors) as compared to their frequency in the overall patient population (~18% of all medulloblastoma tumors contain ecDNA), suggesting ecDNA as a driver for successful establishment of MB PDX models. Single-cell analysis revealed that ecDNA is omnipresent in almost all PDX tumor cells, while its presence was limited to only ~9% of tumor cells in the primary tumor. Conclusion: Analysis of ecDNA in medulloblastoma primary tumors and in PDX models revealed that ecDNA is preserved in PDX models. This is an important characteristic that renders patient-derived mouse xenografts as faithful models of their primary tumors. These results emphasize the relevance of PDX models for preclinical in vivo studies aimed at analyzing the role of ecDNA under therapeutic pressure and for analyzing clonal selection along with molecular evolution of ecDNA during the development of therapeutic resistance. Citation Format: Rishaan Kenkre, Owen Chapman, Jens Luebeck, Vineet Bafna, Robert Wechsler-Reya, Jill Mesirov, Lukas Chavez. Conservation and faithful representation of circular extrachromosomal DNA in orthotopic patient-derived medulloblastoma xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2271.

Targeting the fibroblast growth factor pathway in molecular subtypes of castration-resistant prostate cancer.

Androgen receptor (AR) pathway inhibition remains the cornerstone for prostate cancer therapies. However, castration-resistant prostate cancer (CRPC) tumors can resist AR signaling inhibitors through AR amplification and AR splice variants in AR-positive CRPC (ARPC), and conversion to AR-null phenotypes, such as double-negative prostate cancer (DNPC) and small cell or neuroendocrine prostate cancer (SCNPC). We have shown previously that DNPC can bypass AR-dependence through fibroblast growth factor receptor (FGFR) signaling. However, the role of the FGFR pathway in other CRPC phenotypes has not been elucidated. RNA-Seq analysis was conducted on patient metastases, LuCaP patient-derived xenograft (PDX) models, and CRPC cell lines. Cell lines (C4-2B, VCaP, and 22Rv1) and ex vivo LuCaP PDX tumor cells were treated with enzalutamide (ENZA) and FGFR inhibitors (FGFRi) alone or in combination and sensitivity was determined using cell viability assays. In vivo efficacy of FGFRi in ARPC, DNPC, and SCNPC were evaluated using PDX models. RNA-Seq analysis of FGFR signaling in metastatic specimens, LuCaP PDX models, and CRPC cell lines revealed significant FGF pathway activation in AR-low PC (ARLPC), DNPC, and SCNPC tumors. In vitro/ex vivo analysis of erdafitinib and CH5183284 demonstrated robust and moderate growth suppression of ARPC, respectively. In vivo studies using four ARPC PDX models showed that combination ENZA and CH5183284 significantly suppressed tumor growth. Additional in vivo studies using four ARPC PDX models revealed that erdafitinib monotherapy was as effective as ENZA in suppressing tumor growth, and there was limited combination benefit. Furthermore, two of three DNPC models and two of four SCNPC models responded to CH5183284 monotherapy, suggesting FGFRi responses were model dependent. RNA-Seq and gene set enrichment analysis of end-of-study ARPC tumors treated with FGFRi displayed decreased expression of E2F and MYC target genes and suppressed G2M checkpoint genes, whereas end-of-study SCNPC tumors had heterogeneous transcriptional responses. Although FGFRi treatments suppressed tumor growth across CRPC phenotypes, our analyses did not identify a single pathway or biomarker that would identify tumor response to FGFRi. This is very likely due to the array of FGFR1-4 expression and tumor phenotypes present in CRPC. Nevertheless, our data nominate the FGFR pathway as a clinically actionable target that promotes tumor growth in diverse phenotypes of treatment-refractory metastatic CRPC.

Abstract 1747: Targeting metabolic and epigenetic programs to re-sensitize glioblastoma to chemotherapy

Abstract Treatment options for glioblastoma (GBM) are limited. Prognosis remains dismal, with an 18 month on average survival rate following diagnosis due to treatment resistance and disease recurrence. The goal of this project is to investigate hallmarks of cancer progression that contribute to temozolomide (TMZ) resistance, a first tine treatment for GBM. Two signaling pathways were investigated in TMZ-sensitive and -resistant GBM cell lines and in primary and recurrent patient-derived xenograft (PDX) tumor cells by genetically and pharmacologically inhibiting methionine adenosyltransferase 2A (MAT2A) and adenosylhomocysteinase (AHCY). Cell growth and survival were assessed by measuring protein expression of proliferation, oxidative stress and cell cycle arrest markers. EPIC array analysis and targeted bisulfite sequencing were conducted to identify changes in genome-wide and specific CpG island methylation. The Seahorse XF Analyzer measured mitochondrial respiratory capacity and oxidative metabolism. Induced pluripotent stem cell organoids were co-cultured with PDX tumor cells to determine if treatments mitigate tumor cell invasiveness. Compared to parental cells (PC), MAT2A gene expression was increased by 1.7-fold in acquired resistant and de novo resistant GBM cells (RC) [(transcript per million): PC, 7386 ± 412; RC, 12941 ± 1023; n=2; p=2.10e-8].Compared to TMZ-sensitive cells (TS), TMZ-resistant cells (TR) demonstrated a 56% increase in baseline oxygen consumption rate [(pmol/min): TS, 179 ± 6.7; TR, 279 ± 13; n=18; p=.0012] and 64% increase in maximal respiratory capacity [(pmol/min): TS, 403 ± 29; TR, 659 ± 35; n=6; p<.0001]. Both primary and recurrent GBM cells were subject to select MAT2A and AHCY inhibitor compounds and EC50s were determined. Recurrent cells demonstrated a vulnerability to TMZ-generated cell death both by genetically knocking down and pharmacologically inhibiting MAT2A and AHCY. MAT2A and AHCY contribute to TMZ resistance and recurrence by dysregulating methylation programs and upregulating antioxidant programs, respectively. These findings provide a foundation for developing novel combinatory therapeutic strategies and inform clinical studies intended to augment the practically negligible rate of remission for GBM and improve treatment outcomes for cancer patients. Citation Format: Emma Rowland, Jordan Walter, Anna Jermakowicz, Robert Suter, Rebecca Riggins, Nagi Ayad. Targeting metabolic and epigenetic programs to re-sensitize glioblastoma to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1747.

337 Targeting metabolic and epigenetic programs to re-sensitize glioblastoma to chemotherapy

OBJECTIVES/GOALS: Treatment options for glioblastoma (GBM) are limited. Prognosis remains dismal, with an 18 month on average survival rate following diagnosis due to treatment resistance and disease recurrence. The goal of this project is to investigate hallmarks of cancer progression that contribute to temozolomide (TMZ) resistance, a first tine treatment for GBM. METHODS/STUDY POPULATION: Two signaling pathways were investigated in TMZ-sensitive and -resistant GBM cell lines and in primary and recurrent patient-derived xenograft (PDX) tumor cells by genetically and pharmacologically inhibiting methionine adenosyltransferase 2A (MAT2A) and adenosylhomocysteinase (AHCY). Cell growth and survival were assessed by measuring protein expression of proliferation, oxidative stress and cell cycle arrest markers. EPIC array analysis and targeted bisulfite sequencing were conducted to identify changes in genome-wide and specific CpG island methylation. The Seahorse XF Analyzer measured mitochondrial respiratory capacity and oxidative metabolism. Induced pluripotent stem cell organoids were co-cultured with PDX tumor cells to determine if treatments mitigate tumor cell invasiveness. RESULTS/ANTICIPATED RESULTS: Compared to parental cells (PC), MAT2A gene expression was increased by 1.7-fold in acquired resistant and de novo resistant GBM cells (RC) [(transcript per million): PC, 7386 ± 0.012; RC, 12925 ± 0.023; n=2; p=2.10e-8]. Compared to TMZ-sensitive cells (TS), TMZ-resistant cells (TR) demonstrated a 56% increase in baseline oxygen consumption rate [(pmol/min): TS, 179 ± 6.7; TR, 279 ± 13; n=18; p=.0012] and 64% increase in maximal respiratory capacity [(pmol/min): TS, 403 ± 29; TR, 659 ± 35; n=6; p DISCUSSION/SIGNIFICANCE: MAT2A and AHCY contribute to TMZ resistance and recurrence by dysregulating methylation programs and upregulating antioxidant programs, respectively. These findings provide a foundation for developing novel combinatory therapeutic strategies and inform clinical studies intended to increase remission and reduce recurrence for GBM patients.

Evaluation of Protein Kinase cAMP-Activated Catalytic Subunit Alpha as a Therapeutic Target for Fibrolamellar Carcinoma.

Fibrolamellar carcinoma (FLC) is a rare, difficult-to-treat liver cancer primarily affecting pediatric and adolescent patients, and for which precision medicine approaches have historically not been possible. The DNAJB1-PRKACA gene fusion was identified as a driver of FLC pathogenesis. We aimed to assess whether FLC tumors maintain dependency on this gene fusion and determine if PRKACA is a viable therapeutic target. FLC patient-derived xenograft (PDX) shRNA cell lines were implanted subcutaneously into female NOD-SCID mice and tumors were allowed to develop prior to randomization to doxycycline (to induce knockdown) or control groups. Tumor development was assessed every 2 days. To assess the effect of treatment with novel selective PRKACA small molecule kinase inhibitors, BLU0588 and BLU2864, FLC PDX tumor cells were implanted subcutaneously into NOD-SCID mice and tumors allowed to develop. Mice were randomized to treatment (BLU0588 and BLU2864, orally, once daily) or control groups and tumor size determined as previously. Knockdown of DNAJB1-PRKACA reversed a FLC-specific gene signature and reduced PDX tumor growth in mice compared to the control group. Furthermore, FLC PDX tumor growth was significantly reduced with BLU0588 and BLU2864 treatment vs control (P= .003 and P= .0005, respectively). We demonstrated, using an inducible knockdown and small molecule approaches, that FLC PDX tumors were dependent upon DNAJB1-PRKACA fusion activity. In addition, this study serves as a proof-of-concept that PRKACA is a viable therapeutic target for FLC and warrants further investigation.

Regulation of TORC1 by MAPK Signaling Determines Sensitivity and Acquired Resistance to Trametinib in Pediatric BRAFV600E Brain Tumor Models.

We investigated why three patient-derived xenograft (PDX) childhood BRAFV600E-mutant brain tumor models are highly sensitive to trametinib. Mechanisms of acquired resistance selected in situ, and approaches to prevent resistance were also examined, which may translate to both low-grade glioma (LGG) molecular subtypes. Sensitivity to trametinib [MEK inhibitor (MEKi)] alone or in combination with rapamycin (TORC1 inhibitor), was evaluated in pediatric PDX models. The effect of combined treatment of trametinib with rapamycin on development of trametinib resistance in vivo was examined. PDX tissue and tumor cells from trametinib-resistant xenografts were characterized. In pediatric models TORC1 is activated through ERK-mediated inactivation of the tuberous sclerosis complex (TSC): consequently inhibition of MEK also suppressed TORC1 signaling. Trametinib-induced tumor regression correlated with dual inhibition of MAPK/TORC1 signaling, and decoupling TORC1 regulation from BRAF/MAPK control conferred trametinib resistance. In mice, acquired resistance to trametinib developed within three cycles of therapy in all three PDX models. Resistance to trametinib developed in situ is tumor-cell-intrinsic and the mechanism was tumor line specific. Rapamycin retarded or blocked development of resistance. In these three pediatric BRAF-mutant brain tumors, TORC1 signaling is controlled by the MAPK cascade. Trametinib suppressed both MAPK/TORC1 pathways leading to tumor regression. While low-dose intermittent rapamycin to enhance inhibition of TORC1 only modestly enhanced the antitumor activity of trametinib, it prevented or retarded development of trametinib resistance, suggesting future therapeutic approaches using rapamycin analogs in combination with MEKis that may be therapeutically beneficial in both KIAA1549::BRAF- and BRAFV600E-driven gliomas.

Abstract 1458: Targeting fibroblast growth factor receptors in castration-resistant prostate cancer

Abstract Background: Androgen receptor (AR) pathway inhibition remains the cornerstone for prostate cancer therapies. Although AR signaling inhibitors (ARSI), such as enzalutamide (ENZ) and abiraterone (ABI), extend survival in recurrent and castration-resistant prostate cancer (CRPC), durable complete responses are rare. Bypass mechanisms employed by metastatic CRPC include amplification of AR and AR splice variants in AR-high CRPC (ARPC) and conversion to AR-null phenotypes, such as double-negative prostate cancer (DNPC) and small cell or neuroendocrine prostate cancer (SCNPC). We showed previously that DNPC bypasses AR-dependence through fibroblast growth factor (FGF) signaling. However, the utility of targeting the FGF pathway in other molecular subtypes of CRPC remains to be determined. Methods: RNASeq was conducted on 98 patient metastases, 18 LuCaP patient-derived xenograft (PDX) models and CRPC cell lines. Sensitivity to ARSI and FGFR inhibitors (FGFRi) alone or in combination in cell lines (C4-2B, VCaP, 22Rv1, DU145 and NCIH660) or ex vivo PDX tumor cells (10 ARPC models, 2 DNPC models, and 6 SCNPC models) was measured through CellTitre Glo assays. On-target effects of inhibitors were assessed through immunoblot and qPCR. In vivo efficacy of FGFRi in ARPC and SCNPC was conducted using LuCaP PDX models. Results: Interrogation of FGF pathway activity and FGFR expression using RNASeq and FGF signature scores in CRPC metastases, LuCaP PDX models and CRPC cell lines revealed upregulated FGF pathway activation in AR-null DNPC and SCNPC and in subsets of ARPC. In vitro/ex vivo analysis of FGFRi (erdafitinib, CH5183284 and rogaratinib) demonstrated robust growth suppression in DNPC and moderate growth suppression in SCNPC. In vivo CH5183284 administration recapitulated the growth suppression observed in vitro for SCNPC LuCaP 93 and LuCaP 173.1 but not for SCNPC LuCaP 49. Furthermore, in vitro/ex vivo analysis of AR-expressing CRPC models determined that combination ARSI (ENZ, ABI or darolutamide) with FGFRi was superior to ARSI or FGFRi monotherapies in a subset of models. Immunoblot and qPCR analyses verified that FGFRi-mediated growth suppression occurs through inactivation of the MAPK/MEK and ERK signal transduction cascade in AR-expressing CRPC. Finally, ARPC LuCaP PDX models treated with combination ARSI and FGFRi showed significant tumor growth inhibition compared to control and monotherapy groups in vivo. Analysis of resistance mechanisms to FGFRi therapy are ongoing. Conclusions: The emergence of bypass mechanisms that subvert AR-dependence in mCRPC highlights a need for novel therapeutic strategies. Our data nominate the FGF pathway as an actionable target in diverse phenotypes of treatment-refractory mCRPC. Citation Format: Mark P. Labrecque, Lisha G. Brown, Ilsa M. Coleman, Bryce Lakely, Holly M. Nguyen, Eva Corey, Peter S. Nelson, Colm Morrissey. Targeting fibroblast growth factor receptors in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1458.

Targeted Therapy of TERT-Rearranged Neuroblastoma with BET Bromodomain Inhibitor and Proteasome Inhibitor Combination Therapy.

TERT gene rearrangement with transcriptional superenhancers leads to TERT overexpression and neuroblastoma. No targeted therapy is available for clinical trials in patients with TERT-rearranged neuroblastoma. Anticancer agents exerting the best synergistic anticancer effects with BET bromodomain inhibitors were identified by screening an FDA-approved oncology drug library. The synergistic effects of the BET bromodomain inhibitor OTX015 and the proteasome inhibitor carfilzomib were examined by immunoblot and flow cytometry analysis. The anticancer efficacy of OTX015 and carfilzomib combination therapy was investigated in mice xenografted with TERT-rearranged neuroblastoma cell lines or patient-derived xenograft (PDX) tumor cells, and the role of TERT reduction in the anticancer efficacy was examined through rescue experiments in mice. The BET bromodomain protein BRD4 promoted TERT-rearranged neuroblastoma cell proliferation through upregulating TERT expression. Screening of an approved oncology drug library identified the proteasome inhibitor carfilzomib as the agent exerting the best synergistic anticancer effects with BET bromodomain inhibitors including OTX015. OTX015 and carfilzomib synergistically reduced TERT protein expression, induced endoplasmic reticulum stress, and induced TERT-rearranged neuroblastoma cell apoptosis which was blocked by TERT overexpression and endoplasmic reticulum stress antagonists. In mice xenografted with TERT-rearranged neuroblastoma cell lines or PDX tumor cells, OTX015 and carfilzomib synergistically blocked TERT expression, induced tumor cell apoptosis, suppressed tumor progression, and improved mouse survival, which was largely reversed by forced TERT overexpression. OTX015 and carfilzomib combination therapy is likely to be translated into the first clinical trial of a targeted therapy in patients with TERT-rearranged neuroblastoma.

Interrogation of Dysregulated Pathways Enables Precision Medicine in Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy that is initially responsive but ultimately relapses after frontline therapy. Although the first-in-class Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has achieved a 68% overall response rate in relapsed/refractory MCL patients, most experience disease progression after ibrutinib treatment. Furthermore, the diverse heterogeneity of molecular alterations in each patient makes improving patient outcome with a uniform regimen extremely challenging. Therefore, identification of effective drug therapies, especially personalized therapeutic strategies are urgently needed. In this study, clinical patient samples were submitted for molecular profiling analysis to identify the dysregulated pathways for each patient. Isolated tumor cells underwent an in vitro drug screen with a panel of clinic drug candidates. Final in vivo efficacy evaluation on the patient derived xenograft (PDX) mouse models could be utilized to predict and validate the clinical response. Methods: We collected fresh peripheral blood specimens, surgical biopsies, bone marrow aspirates and apheresis samples under established IRB-approved protocols. The extracted tumor RNA was subjected to a CLIA-validated nanoString nCounter analysis to interrogate the dysregulated signaling pathways, and whole-exome sequencing (WES) was conducted to reveal the somatic mutations and DNA copy number alterations. High throughput cell viability assays of 23 clinical drug agents targeting multiple pathways associated with MCL pathogenesis were tested per patient sample using the CellTiter-Glo luminescent assay (Promega). Meanwhile, subcutaneous, intravenous and subrenal injections of the purified patient tumor cells were performed on NSG-mice to create corresponding PDX mouse models and these models were used for in vivo validation of rational therapeutic treatment options for precision medicine. Results: We collected and submitted 21 clinical patient samples for molecular profiling analysis and screened them through the designed drug panel. We identified correlations between the WES and nanoString nCounter analysis to interrogate the dysregulated pathways for each patient. The PI3K/mTOR signaling, cell cycle regulation, and apoptosis pathways were among the three most markedly enriched pathways in the relapsed MCL samples based on the hallmark gene set analysis. For example, aberrant apoptosis pathway was identified as the predominant cancer hallmark in one of the patients, the Bcl-2 inhibitor venetoclax and MCL-1 inhibitor AZD5991 were chosen as rational therapeutic treatment options. Indeed, both venetoclax and AZD5991 dramatically induced cell death in both primary patient cells and isolated PDX tumor cells ex vivo. Furthermore, administration of venetoclax at 50mg/kg eradicated the tumor growth in the PDX mouse model established from this patient (p<0.001). In another patient, dysregulated cell cycle pathway (E2F targets and G2M checkpoint) was the predominant cancer hallmark and treatment with CDK4/6 inhibitor abemaciclib (75mg/kg) significantly decreased the tumor size in the PDX model derived from this patient (p<0.0001). Conclusions: Interrogation of dysregulated pathways could be achieved by molecular profiling analysis, and suggest rational treatment options. Follow-up in vitro & in vivo drug efficacy determination facilitates the identification of potential therapeutic options for precision medicine in MCL patient to ultimately improve patient survival outcome. Disclosures Wang: AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Molecular Templates: Research Funding; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; OncLive: Honoraria; Nobel Insights: Consultancy; InnoCare: Consultancy; Lu Daopei Medical Group: Honoraria; Beijing Medical Award Foundation: Honoraria; Guidepoint Global: Consultancy; Pulse Biosciences: Consultancy; Oncternal: Consultancy, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; MoreHealth: Consultancy; VelosBio: Research Funding; BioInvent: Research Funding; Juno: Consultancy, Research Funding; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Acerta Pharma: Research Funding; Verastem: Research Funding; Loxo Oncology: Consultancy, Research Funding; Targeted Oncology: Honoraria; Dava Oncology: Honoraria; OMI: Honoraria, Other: Travel, accommodation, expenses.

Abstract 2901: Clonal dynamics and phenotypic evolution during chemoresistance and metastasis revealed by patient-derived xenograft models of triple negative breast cancer

Abstract Half of all triple negative breast cancer (TNBC) patients harbor significant residual cancer burden following standard neoadjuvant chemotherapy treatment, resulting in distant metastasis and death for most of these patients. Intra-tumor heterogeneity (ITH) is pervasive in TNBC as is a barrier to development of effective therapeutic strategies, but the relative contributions of heterogeneous tumor cell populations to chemoresistance and metastasis are not well understood. To investigate the clonal dynamics that accompany chemotherapy treatment and metastasis, we employed orthotopic patient-derived xenograft (PDX) models of treatment-naïve TNBC, thus enabling experimentation with heterogeneous populations of human tumor cells that have undergone minimal manipulation. To monitor the fates of PDX tumor cell lineages as they metastasized, we introduced a pooled lentiviral barcode library (Cellecta) into freshly dissociated PDX tumor cells which were orthotopically engrafted into recipient mice. Genomic analyses, including barcode enumeration, whole-exome sequencing, custom targeted DNA sequencing, and transcriptome sequencing, were conducted to characterize the clonal dynamics that accompanied metastasis. Of the thousands of diverse primary tumor clones, only ~2% harbored metastatic capacity. Of those, a rare population of the exact same clones predominated metastases in lung, liver, and brain, the three most common sites of human TNBC metastasis. These studies provide a quantitative map of the clonal architecture of multi-organ metastasis in TNBC and reveal that identical subclones can thrive in diverse secondary organ microenvironments. NACT resistance leads to metastasis and death for most patients, yet the origins of chemoresistance in TNBC are unclear. We modeled NACT resistance in an array of PDX models derived from treatment-naïve TNBC biopsies in alignment with an ongoing neoadjuvant clinical trial (NCT02276443). Upon partial response to NACT, tumors entered a transient drug-tolerant state characterized by distinct histologic, proteomic, and transcriptomic features that were reverted as tumors regrew after cessation of treatment. Barcode-mediated lineage tracking and whole-exome sequencing revealed that the drug-tolerant state was not mediated by clonal selection. Based on transcriptomic and metabolic features of the drug-tolerant state, we conducted preclinical trials with an inhibitor of mitochondrial oxidative phosphorylation (IACS-010759), which significantly delayed the regrowth of residual tumors in PDX models. Together, these studies revealed that TNBCs can resist NACT through non-selective adaptation of a reversible phenotypic state, and that inhibition of oxidative phosphorylation may be a promising therapy in the neoadjuvant setting for TNBC. Citation Format: Gloria V. Echeverria, Zhongqi Ge, Sahil Seth, Emily Powell, Xiaomei Zhang, Sabrina Jeter-Jones, Xinhui Zhou, Yan Jiang, Aaron McCoy, Shirong Cai, Yizheng Tu, Michael Peoples, Yuting Sun, Huan Qiu, Christopher Bristow, Alessandro Carugo, Jiansu Shao, Stacy L. Moulder, William F. Symmans, Timothy P. Heffernan, Jeffrey T. Chang, Helen M. Piwnica-Worms. Clonal dynamics and phenotypic evolution during chemoresistance and metastasis revealed by patient-derived xenograft models of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2901.

Abstract GS5-05: Resistance to neoadjuvant chemotherapy in triple negative breast cancer mediated by a reversible drug-tolerant state

Abstract Approximately 50% of patients with localized triple negative breast cancer (TNBC) have substantial residual cancer burden following treatment with neoadjuvant chemotherapy (NACT), resulting in distant metastasis and death for most of these patients. While genomic and phenotypic intra-tumor heterogeneity are pervasive features of TNBCs at the time of diagnosis, the functional contributions of heterogeneous tumor cell populations to chemoresistance have not been elucidated. To investigate tumor evolution accompanying NACT, we employed orthotopic patient-derived xenograft (PDX) models of treatment-naïve TNBC, which retain intra-tumor heterogeneity characteristic of human TNBC. We discovered that some PDX models initially exhibited partial sensitivity to standard front-line NACT (Adriamycin plus Cytoxan, AC). Following AC, residual tumors were resistant to chemotherapy but repopulated tumors with chemo-sensitive cells if left untreated, indicating that tumor cells possessed inherent plasticity. To identify the tumor cell subpopulation(s) conferring chemoresistance, we conducted barcode-mediated clonal tracking in three independent PDX models by introducing a high-complexity pooled lentiviral barcode library into PDX tumor cells which were then orthotopically engrafted into recipient mice. Strikingly, residual tumors maintained the same heterogeneous clonal architecture as naïve tumors. Concordantly, whole-exome sequencing revealed conservation of genomic subclonal architecture throughout treatment. These results were corroborated by genomic sequencing of serial biopsies pre- and post-AC obtained directly from TNBC patients enrolled on an ongoing clinical trial at MD Anderson (ARTEMIS; NCT02276443). Together, these studies revealed that genomically distinct pre-treatment subclones were equally capable of surviving AC to reconstitute tumors after treatment. To identify functional addictions of residual tumor cells, we conducted histologic and transcriptomic profiling. Residual tumors following AC-treatment exhibited extensive fibrotic desmoplasia and tumor cell pleomorphism in both PDX models and in serial biopsies obtained from TNBC patients enrolled on the ARTEMIS trial. Strikingly, these AC-induced features were reverted upon regrowth of residual tumors in PDXs and in patients' tumors. Similarly, residual tumors exhibited unique transcriptomic features, many of which are also de-regulated in cohorts of human TNBCs undergoing chemotherapy treatment. These features were nearly completely reverted after tumors regrew, suggesting that the residual tumor state may be a unique and transient therapeutic window. Gene set enrichment analyses revealed that residual tumors had increased activation of oxidative phosphorylation and decreased glycolytic signaling. Pharmacologic targeting of oxidative phosphorylation with a small-molecule inhibitor of mitochondrial electron transport chain complex I (IACS-010759) significantly delayed the regrowth of AC-treated residual tumors in three independent PDX models. Collectively, these studies reveal that a reversible phenotypic state can confer chemoresistance in the absence of genomic selection and that the residual tumor state is a novel therapeutic window for chemo-refractory TNBC. Citation Format: Echeverria GV, Ge Z, Seth S, Jeter-Jones SL, Zhang X, Zhou X, Cai S, Tu Y, McCoy A, Peoples M, Lau R, Shao J, Sun Y, Bristow C, Carugo A, Ma X, Harris A, Wu Y, Moulder S, Symmans WF, Marszalek JR, Heffernan TP, Chang JT, Piwnica-Worms H. Resistance to neoadjuvant chemotherapy in triple negative breast cancer mediated by a reversible drug-tolerant state [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr GS5-05.

Abstract 212: High-resolution barcoding in patient-derived xenografts of triple-negative breast cancer reveals reversible chemoresistance conferred by non-mutational mechanisms

Abstract Fifty percent of patients with localized triple negative breast cancer (TNBC) have substantial residual cancer burden following treatment with neoadjuvant chemotherapy (NACT), resulting in a 40-80% risk of recurrence which leads to distant metastases and death for most patients. Intra-tumor heterogeneity (ITH) is a pervasive feature of TNBCs but the relative contributions of heterogeneous tumor cell populations to chemoresistance are not understood. To investigate the clonal dynamics that accompany chemotherapy treatment, we employed orthotopic patient-derived xenograft (PDX) models of treatment-naïve TNBC, thus enabling experimentation with heterogeneous populations of human tumor cells that have undergone minimal manipulation. To monitor the fates of PDX tumor cell lineages undergoing treatment with front-line NACT, we treated multiple treatment-naïve PDX models with Adriamycin combined with Cytoxan (AC). Some PDXs initially exhibited partial sensitivity followed by maintenance of residual tumors that were resistant to chemotherapy. Residual tumors re-grew to regain partial chemo-sensitivity. To conduct barcode-mediated clonal tracking, we introduced a pooled lentiviral barcode library (Cellecta; 50M unique barcodes) into freshly dissociated PDX tumor cells which were orthotopically engrafted into recipient mice. Strikingly, residual tumors maintained the same clonal architecture as untreated tumors. In contrast, only 20% of residual tumor clones repopulated tumors after discontinuation of treatment. Whole-exome sequencing revealed conservation of mutant allele frequencies throughout treatment. Together, these studies demonstrate that re-growth of residual tumors is accompanied by a non-random selection of subclones, that residual tumors surviving AC maintain the same levels of ITH as untreated tumors, and that selection of genomic subclones did not confer the observed chemoresistance. Transcriptomic, proteomic, and histologic profiling revealed that residual tumors exist in a distinct state characterized by alterations in EMT, metabolic, and cell adhesion programs. This state was reverted as tumors re-grew after discontinuation of AC treatment, indicating that the residual state may be a unique therapeutic window. In silico prediction of drug sensitivity revealed candidate drivers of resistance in the residual tumor state, and pharmacologic targeting identified multiple existing therapies that significantly delayed the regrowth of residual tumors. These data suggest that sequential administration of AC followed by these targeted agents could prolong TNBC responses, which may delay time to recurrence for patients with this highly aggressive disease. Citation Format: Gloria Vittone Echeverria, Sahil Seth, Zhongqi Ge, Alessandro Carugo, Christopher Bristow, Prabjhot Mundi, Sabrina Jeter-Jones, Xiaomei Zhang, Xinhui Zhou, Aaron McCoy, Shirong Cai, Yizheng Tu, Yuting Sun, Joseph Marszalek, Andrea Califano, William F. Symmans, Stacy L. Moulder, Jeffery T. Chang, Timothy P. Heffernan, Helen Piwnica-Worms. High-resolution barcoding in patient-derived xenografts of triple-negative breast cancer reveals reversible chemoresistance conferred by non-mutational mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 212.

TMOD-10. INTRACRANIAL PATIENT-DERIVED XENOGRAFT FROM A PEDIATRIC GLIOBLASTOMA RETAINS MOLECULAR CHARACTERISTICS AND GROWTH PATHWAYS

BACKGROUND: Pediatric glioblastomas (pGBM) are characterized by overlapping and unique molecular driver mutations compared to their adult counterparts. Primary cell cultures and patient-derived xenograft (PDX) models of pGBM are rare which may contribute to difficulty in advancing patient outcomes. We have established an orthotopic PDX mouse model from a pGBM resected from a 12 yo male with recurrent disease. The patient received focal radiation and several chemotherapy regimens prior to the recurrence leading to this tumor resection. METHODS: The surgical tumor specimen was dissociated and directly implanted into the brains of immunocompromised (NSG) mice. DNA and immunohistochemical analyses of the patient tumor and PDX were performed. Multiple in vitro culture conditions were tested on the dissociated primary tumor cells and PDX tumor cells. RESULTS: Implantation of the patient tumor cells in mice resulted in tumor growth after 120–250 days. Tumors were serially transplanted successfully. Attempts to establish in vitro cell cultures from primary tumor and from established PDX tumors failed. DNA sequencing of the patient tumor sample and tumors from each PDX generation revealed the H3F3A G34R mutation characteristic of cortical pGBM, and mutations in the TP53, PTEN and PIK3CA genes, all of which were conserved in PDX tumors. Interestingly, the patient tumor showed evidence of telomerase activity and alternative lengthening of telomeres (ALT) as did the PDX tumors. The combined PTEN and PIK3CA mutations in this tumor resulted in activation of the PI3K-mTOR pathway. Treatment of tumor-bearing mice with an mTOR or PI3K inhibitor resulted in suppression of the pathway and reduced proliferation. CONCLUSION: This pGBM PDX model conserved key driver mutations and pathways from the patient tumor. In vitro culture of pGBM can be extremely challenging to establish and more difficult methods such as orthotopic PDX may be required to study these tumors.

Pre-Clinical Evaluation of the PI3K-p110β/δ Inhibitor KA2237 in Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) accounts for 6% of all non-Hodgkin lymphoma and is a therapeutic challenge. Phosphoinositide-3 kinase (PI3K) has been shown to be an alternative survival pathway in relapsed/refractory MCL. KA2237 (designed by Karus Therapeutics Ltd, Oxfordshire, United Kingdom) is a dual inhibitor of the class I beta and delta isoforms of the 110 kDa catalytic subunit of PI3K. By selectively targeting PI3K-beta and -delta isoforms and preventing their activation, KA2237 may decrease proliferation and induce cell death in susceptible tumor cells.Methods: We assessed the effects of KA2237 on the in vitro cell proliferation of both ibrutinib-sensitive (Mino, Jeko-1, and Rec-1) and primary ibrutinib-resistant (Z-138 and Maver-1) cell lines, and acquired ibrutinib-resistant MCL cell line, Jeko-R. We also tested the viability of patient-derived xenograft (PDX) tumor cells to KA2237. We compared the efficacy of KA2237 with two other commercial PI3K inhibitors, duvelisib (IPI-145, Selleck) and idelalisib (Cal-101, Selleck). Also, we paired these three inhibitors (KA2237, duvelisib and idelalisib) each with ibrutinib to evaluate the potential synergistic effects of these combinations. Lastly, we also tested in vivo efficacy of KA2237 and its combination with ibrutinib in PDX tumor cells.Results: KA2237 inhibited cell proliferation in both ibrutinib-sensitive and ibrutinib-resistant cell lines in a dose-dependent and time-dependent manner. For Mino and Jeko-1, the IC50 was 4.8 uM and 2.9 uM and for Z-138 and Maver-1 cell lines, the IC50 was 0.6 uM and 0.1 uM, respectively. KA2237 also decreased cell viability of ibrutinib-sensitive and ibrutinib-resistant MCL PDX tumor cells. However, KA2237 did not decrease the cell viability of normal human peripheral blood mono-nuclear cells. KA2237 arrested phase G0/G1 in Rec-1 and Jeko-R cell lines. We detected the expression of PI3K isoforms in MCL, finding higher expression of PI3K β and δ in MCL-resistant cell lines as compared with sensitive cell lines. We found that KA2237 induced MCL cell apoptosis in a time-dependent and dose-dependent manner. In comparison with duvelisib and idelalisib, KA2237 achieved greater inhibition of cell viability, cell apoptosis and cell cycle arrest. Furthermore, we found synergistic effects of KA2237 and ibrutinib combination in several MCL cell lines and in PDX models. In an ibrutinib-resistant PDX model, KA2237 treated mice reduced tumor burden significantly compared with vehicle control, and higher tumor growth inhibition was achieved as compared with ibrutinib.Conclusion: The novel PI3K inhibitor, KA2237 may be a potential candidate for MCL therapy, especially in the ibrutinib-resistant cases. DisclosuresWang:Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Research Funding; Pharmacyclics: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Research Funding; BeiGene: Research Funding; Asana BioSciences: Research Funding; Kite Pharma: Research Funding; Celgene: Research Funding.

Patient-Derived Xenograft Model in B-Cell Lymphoma: A Platform for a Personalized Clinical Trial

Background: Imprecise correlation of treatment response in established cell line and cells obtained from patients (i.e., patient-primary) limits the preclinical investigation of novel compounds. Similarly, the "xenograft" models wherein human cancer cell lines are transplanted into immunocompromised mice do not represent the full spectrum of cancers. However, Patient-derived xenograft (PDX) mouse models have been shown to recapitulate the diversity of growth, metastasis, and histopathology of the original tumor. Based on our previously established mantle cell lymphoma (MCL) PDX model, we developed other B-cell lymphoma PDXs recapitulating tumor pathological and clinical characteristics, progression and response to therapeutic agents, this will provide an indispensable model system towards personalized treatment for B-cell lymphoma.Methods: We developed 34 PDX models with an implanted fetal bone chip for several B-cell lymphomas including marginal zone lymphoma (MZL), follicular lymphoma (FL), Burkitt’s lymphoma (BL), and diffuse large B-cell lymphoma (DLBCL), and MCL. We tested the in vitro efficacy of a panel of drugs among freshly isolated tumor cells from patients and tumor cells from the PDX models. We also generated a drug-resistant MCL PDX model, compared the effect of targeted drugs on the tumor burden in the drug-resistant model and identified potential therapeutic opportunity with drug combinations. We validated combination therapy in vivoand conducted next generation sequencing (NGS) using a 1,212 gene panel (OncoPlus®) on DNA from primary patient cells.Results: We collected clinical samples from 34 patients with several types of B-cell lymphomas including MCL (n=21), DLBCL (n=3), FL (n=2), BL (n=1), and MZL (n=2). Of the 34 patients, 18 (53%) were newly diagnosed and untreated clinically, 11 (32%) patients were relapsed after treatment with 1-3 chemotherapy or targeted therapy treatments, and 5 (15%) patients were treated with one-dose therapy before sample collection. All of the tumor cells, from both the patient and PDXs, showed the same drug response pattern. Consecutive ibrutinib administration to PDX mice from PT1 during G4 induced the development of an ibrutinib-resistant tumor. The cell viability of isolated PDX tumor cells treated with ibrutinib was not significantly different between G1 and G2 nor was it different between G5 and G6 (p>0.05). In addition, histological features were consistent with patient tumor histology. Furthermore, whole exome sequencing revealed fidelity between the patient and PDX tumor cells as well as between subsequent generations of the PDX. [B1] We[Z2] engrafted a patients tumor cells into NSG-hu mice to create an MCL-bearing PDX mouse model (PT28-PDX). G2 PDX cells were isolated and treated with a panel of drugs. We found that G2 PDX cells were most sensitive to bortezomib (BTZ) (Velcade®). Growth inhibition of the G2 PDX cells was significantly higher with BTZ compared to ibrutinib (p=0.002) and cells were most sensitive to BTZ compared with other agents (p≤ 0.002). Based on this finding, the patient was treated with Velcade®, rituximab and dexamethasone and responded to treatment. However, the G3 PDX became resistant to BTZ. She was then treated with a 1 cycle of rituximab and cytarabine. Soon after, we initiated three-drug combination treatment with lenalidomide (Len), rituximab (RTX) and dexamethasone (DEX) in PDX G4. Len+RTX+DEX significantly prolonged mouse survival indicating that this could be an effective regimen for this patient after BTZ relapse. As guided by the PDX, PT28 underwent Len+RTX+DEX regimen and her peripheral lymphocytosis disappeared demonstrating response.Conclusions: The PDX model with implanted fetal bone chip is a valid experimental platform that recapitulates tumor characteristics of B-cell lymphomas. Leveraging the PDX platform to identify and select drugs that are likely to be efficacious for individual patients and subsequently administering the promising agents to those who relapse after an initial therapy is the next milestone. DisclosuresWang:BeiGene: Research Funding; Asana BioSciences: Research Funding; Juno Therapeutics: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding; Onyx: Research Funding; Kite Pharma: Research Funding; Acerta Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract B39: Generation of head and neck cancer patient-derived xenografts with in vivo acquired cetuximab resistance

Abstract Background: Although cetuximab has demonstrated only modest response rates in head and neck squamous cell carcinoma (HNSCC) patients in clinical studies, most HNSCC cell line xenograft models have demonstrated sensitivity to cetuximab. We sought to characterize molecular and phenotypic changes associated with the acquisition of cetuximab resistance by evaluating patient-derived xenograft (PDX) models with and without cetuximab selection in vivo. Methods: A single PDX was established from a patient tongue HNSCC and a separate PDX from an independent patient lymph node by subcutaneous propagation in nod scid gamma (NSG) immunodeficient mice. Established PDXs were implanted into 2 flanks of NSG mice, and mice were either untreated (parental) or treated with cetuximab using modulated dosing dependent upon tumor growth (0.04-0.2 mg 1-2 times weekly) with the goal of PDX growth at the maximum dose. Tumor histology and short tandem repeat (STR) profiling were evaluated for HNSCC tumors and paired PDX models. Cell lines were derived from PDX tumors grown in NSG hypoxanthine-guanine phosphoribosyltransferase (Hprt)-deficient mice, allowing negative selection of mouse fibroblasts in vitro. Cell lines were retrovirally transduced to express either green fluorescent protein (GFP) or telomerase (HTERT). Cell lines were evaluated for presence of human cells using a PCR-based assay to detect human amelogenin alleles (sex id assay). Candidate gene mutations and protein/phosphoprotein levels will be evaluated in parental and cetuximab-selected PDXs using Ion Torrent and immunoblotting technologies, respectively. Results: Patient HNSCC tumors and paired parental PDXs displayed similar histologies and consistent STR profiles. Four of 14 PDXs derived from the tongue HNSCC displayed growth in the presence of cetuximab at the maximum dose, and of 18 PDXs derived from lymph node, 3 grew in the presence of cetuximab at the maximum dose. Average tumor volume at treatment initiation was 134 mm3 and average duration of treatment was 8 months. For tumors that eventually grew in presence of maximum dose cetuximab, initial tumor volume and duration of treatment were 156.5 mm3 and 9 months, respectively. Cetuximab-resistant tumors were heterogeneous regarding their resistance to cetuximab in a subsequent passage. Of the two cetuximab-resistant tumors STR evaluated, 1 was consistent with the patient and parental PDX and 1 could not be genotyped. In vitro cell cultures were established from parental PDX tumor cells transduced with HTERT and GFP, and these cells were confirmed to be of human origin by the sex id assay. Molecular profiling and establishment of cell lines derived from cetuximab-resistant, genotype confirmed PDXs are currently ongoing. Conclusions: It was possible to obtain PDX tumors grown under conditions of chronic treatment with cetuximab. However, dose modulation was required and propagation of the trait of cetuximab-resistant growth upon PDX passage was not guaranteed. The acquisition of cetuximab-resistant PDX tumors was a lengthy process that required care regarding dosing modulation and genotype validation. Once established, these models will provide valuable insights regarding molecular and phenotypic changes associated with acquired cetuximab resistance. Citation Format: Pei Zhou, Hua Li, Sarah Wheeler, Jennifer R. Grandis, Laura A. Stabile, Ann Marie Egloff. Generation of head and neck cancer patient-derived xenografts with in vivo acquired cetuximab resistance. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B39.